A new class of nuclear antigen for the Epstein-Barr virus (EBV) which we term Class II EBNA has been identified. This EBNA differs from the classical antigen (we term Class I EBNA) by immunological and biochemical properties. It is possible that the Class II EBNA is the tool by which the EBV infects and transforms human lymphocytes. Using specific sera to detect only viral related proteins, the Class I and Class II EBNA were shown to involve different antigens/antibodies. The Class II antigen is more basic than the Class I antigen monomer but is not a histone. It is bound more tightly to DNA than Class I EBNA or histones. It can be separated from histones and Class I EBNA by hydroxylapatite chromatography, isoelectric focusing and by two-dimensional polyacrylamide gel electrophoresis. This new antigen is only found in infected (positive) cells and only with positive sera; therefore, it is definitely EBV related. Purification of this Class II antigen is underway using ongoing techniques in the laboratory for fractionating nonhistone chromosomal proteins, e.g. preparative isoelectric focusing, hydrophobic and adsorption chromatography, DNA affinity chromatography, etc. I plan to finalize the purification of this Class II EBNA and chemically characterize it including its primary sequence. It is hoped to use the primary sequence of the both Class I and Class II protein antigens to generate synthetic DNA probes coding for the sequences in the N terminal and, hopefully, the middle and C terminal regions of the protein. These probes would then be used to identify either host cell genomic fractions (human) or the viral genome as containing the gene for Class I and II EBNA. Isolation of genomic regions containing the EBNA genes are possible. I also plan a major effort to prepare monoclonal antibodies against the Class II EBNA. The antibodies will be used to compare soluble, Class I and Class II EBNA's antigenically, to provide an alternate method of purifying the antigens, to probe the appearance of these antigens after EBV infection, and to determine the intranuclear location of the EBNA. The antibodies will also be used to help isolate nuclease digested DNA fragments of the intact Class II EBNA-DNA complexes for enrichment of the DNA sequences bound by this antigen.